U.S. patent number 11,446,235 [Application Number 16/609,346] was granted by the patent office on 2022-09-20 for cosmetic compositions for skin health and methods of using same.
This patent grant is currently assigned to LOCUS IP COMPANY, LLC. The grantee listed for this patent is Locus IP Company, LLC. Invention is credited to Ken Alibek, Sean Farmer, Sharmistha Mazumder.
United States Patent |
11,446,235 |
Farmer , et al. |
September 20, 2022 |
Cosmetic compositions for skin health and methods of using same
Abstract
The topical cosmetic compositions and methods of the subject
invention can be used to treat and/or prevent a variety of skin
conditions, including, for example, age spots, acne, scars, body
odor, aging-related conditions (e.g., wrinkles, looseness and
dryness), and scalp issues (e.g., dandruff, seborrheic dermatitis
and hair loss). In preferred embodiments, the compositions
according to the subject invention comprise biological amphiphilic
molecules produced by microorganisms.
Inventors: |
Farmer; Sean (North Miami
Beach, FL), Alibek; Ken (Solon, OH), Mazumder;
Sharmistha (Copley, OH) |
Applicant: |
Name |
City |
State |
Country |
Type |
Locus IP Company, LLC |
Solon |
OH |
US |
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Assignee: |
LOCUS IP COMPANY, LLC (Solon,
OH)
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Family
ID: |
1000006573363 |
Appl.
No.: |
16/609,346 |
Filed: |
April 30, 2018 |
PCT
Filed: |
April 30, 2018 |
PCT No.: |
PCT/US2018/030229 |
371(c)(1),(2),(4) Date: |
October 29, 2019 |
PCT
Pub. No.: |
WO2018/208530 |
PCT
Pub. Date: |
November 15, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200069779 A1 |
Mar 5, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62502714 |
May 7, 2017 |
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62537057 |
Jul 26, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61Q
15/00 (20130101); A61K 9/0014 (20130101); A61P
17/02 (20180101); A61K 8/8147 (20130101); A61P
17/10 (20180101); A61K 8/99 (20130101); A61Q
19/08 (20130101); A61K 36/062 (20130101); A61K
45/06 (20130101); A61K 8/735 (20130101); A61K
9/06 (20130101); A61Q 19/00 (20130101); A61Q
19/007 (20130101); A61Q 19/008 (20130101); A61K
8/66 (20130101); A61K 31/05 (20130101); A61K
8/347 (20130101); A61K 9/12 (20130101); A61Q
19/02 (20130101); A61K 38/47 (20130101); A61Q
17/00 (20130101); A61K 8/9794 (20170801); A61K
35/74 (20130101); A61K 31/728 (20130101); A61K
9/08 (20130101); A61Q 5/00 (20130101); A61K
9/107 (20130101); A61K 9/10 (20130101); A61K
8/64 (20130101) |
Current International
Class: |
A61K
8/99 (20170101); A61K 9/08 (20060101); A61K
9/10 (20060101); A61K 9/107 (20060101); A61K
9/12 (20060101); A61K 31/05 (20060101); A61K
31/728 (20060101); A61K 35/74 (20150101); A61K
36/062 (20060101); A61K 38/47 (20060101); A61K
45/06 (20060101); A61P 17/02 (20060101); A61P
17/10 (20060101); A61Q 5/00 (20060101); A61Q
15/00 (20060101); A61K 9/06 (20060101); A61K
9/00 (20060101); A61K 8/9794 (20170101); A61K
8/81 (20060101); A61K 8/73 (20060101); A61K
8/66 (20060101); A61K 8/64 (20060101); A61K
8/34 (20060101); A61Q 19/02 (20060101); A61Q
17/00 (20060101); A61Q 19/00 (20060101); A61Q
19/08 (20060101) |
References Cited
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Other References
De Brito, D., Biosurfactants from renewable raw materials,
Universidade do Minho Departamento de Engenharia Biologica, Nov.
2013, pp. 1-93. cited by applicant .
De Oliveira, M., et al., "Review: Sophorolipids A Promising
Biosurfactant and it's Applications." International Journal of
Advanced Biotechnology and Research, 2015, 6(2): 161-174. cited by
applicant .
Gharaei-Fathabad, E., "Biosurfactants in Pharmaceutical Industry (A
Mini-Review)." American Journal of Drug Discovery and Development,
2010, 1(1): 58-69. cited by applicant .
Kurtzman, C.P., et al., "Production of sophorolipid biosurfactants
by multiple species of the Starmerella (Candida) bombicolayeast
clade." FEMS Microbiol Lett, 2010, 311: 140-146. cited by applicant
.
Nitschke, M., et al., "Production and properties of a surfactant
obtained from Bacillus subtilis grown on cassava wastewater."
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Molecular Sciences, 2016, 17(401): 1-31. cited by applicant .
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Biology." Landes Bioscience and Springer Science +Business Media,
LLC, 2010, 672: 1-331. cited by applicant .
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Lactobacillus and Bacillus sp." Int. J. Curr. Microbiol. App. Sci.,
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biosurfactant di-rhamnolipid suggests a therapy against scar
formation " Scientific Reports, 2016, 6: 37553, pp. 1-10. cited by
applicant .
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.
Takahashi, M., et al., "Production of Sophorolipid Glycolipid
Biosurfactants from Sugarcane Molasses Using Starmerella bombicola
NBRC 10243." Journal of Oleo Science, 2011, 60(5): 267-273. cited
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Torres Faria, N., et al., "Production of glycolipid biosurfactants,
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Primary Examiner: Lee; Sin J
Attorney, Agent or Firm: Saliwanchik, Lloyd &
Eisenschenk
Parent Case Text
This application is a National Stage Application of International
Application No. PCT/US2018/030229, filed Apr. 30, 2018; which
claims the benefit of the following U.S. provisional applications:
Ser. No. 62/502,714, filed May 7, 2017; and Ser. No. 62/537,057,
filed Jul. 26, 2017, each of which is incorporated herein by
reference in its entirety.
Claims
We claim:
1. A method for treating actinic keratosis, wherein the method
comprises applying a composition to a skin of a human in need of
such treatment, wherein said composition comprises a
therapeutically effective amount of a rhamnolipid and a
topically-acceptable vehicle.
2. The method of claim 1, wherein said rhamnolipid is produced by
Pseudomonas bacteria.
3. The method of claim 1, wherein the composition is applied once
or twice daily.
4. The method of claim 1, further comprising rinsing the
composition from the skin.
5. The method of claim 1, wherein said composition further
comprises a therapeutically effective amount of resveratrol,
hyaluronic acid and/or polyacrylic acid.
Description
BACKGROUND OF THE INVENTION
The skin, or integument, is the largest organ of the human body. It
is comprised of layers, including the epidermis, dermis, and
hypodermis. Due, in part, to the external nature of the skin and
its exposure to a panoply of environmental agents, the skin can be
affected by a wide variety of conditions--some of which are more
serious than others.
One skin condition, actinic keratoses (commonly known as age spots
or liver spots), comprises flat, tan-, brown- or black-colored
spots on the skin. The spots can vary in size and usually appear on
the face, hands, shoulders and arms. While age spots are sometimes
referred to as liver spots, their cause does not actually relate to
liver problems. Instead, age spots are the result of excess
production of the pigment melanin.
There is no definitive opinion as to why age spots develop.
Currently, however, it is widely accepted that skin aging, sun
exposure, or other forms of ultraviolet (UV) light exposure, such
as tanning beds, can be possible causes. Furthermore, some research
suggests that other factors such as viral and fungal infectious
agents, immune changes in the skin, and oxidative stress caused by
a variety of ROS (reactive oxygen species) molecules can play a
role in the causation of keratoses.
The coloration of age spots ranges from light brown to black.
Typically, their texture resembles that of the rest of the skin,
and they are located on sun-exposed areas of the skin. Sometimes,
the spots can have more dominant structure and can look "hilly"
over the normal skin surface. In many cases, age spots are
painless, pose no danger and cause no health problems. Some age
spots can develop into skin cancer, however. For this reason, as
well as their undesirable appearance, many people seek to have age
spots removed.
Clinically, actinic keratoses present as erythematous keratotic
macules, papules, and plaques. Most patients have multiple spots or
lesions, and will continue to develop new lesions as a result of,
for example, past UV-induced DNA damage, as well as cutaneous
immunosuppression caused by continuing UV exposure.
Histologically, actinic keratoses are observed as an
intraepithelial proliferation of abnormal keratinocytes. If these
cells extend beyond the basement membrane, a spot can become
malignant and transform into invasive squamous cell carcinoma.
The most aggressive form of actinic keratosis is keratotinic
cheilitis, which appears on the lips and often transforms into
squamous cell carcinoma. Squamous cell carcinoma and actinic
keratosis share epidemiologic, cytologic, and molecular features.
Epidemiologic data indicate that at least 10% of actinic keratosis
lesions may progress to squamous cell carcinoma within 10 years,
and 60% of squamous cell carcinomas arise from clinically diagnosed
actinic keratoses. Individuals who are immunosuppressed as a result
of cancer, chemotherapy, AIDS, or organ transplantation are also at
a higher risk.
Another form of keratosis, seborrheic keratosis (SK), is a skin
condition that results from a common skin growth. SK is amongst the
most common cutaneous lesions, appearing in middle-aged and older
adults, and affecting some 83 million Americans. This type of
keratosis is mostly benign, but in very rare cases there have been
reports of melanoma development within a seborrheic wart.
Seborrheic keratoses can develop almost anywhere on the skin,
though most commonly on the neck, face and trunk. The growths are
often round or oval shaped, and appear as flat or slightly raised
spots on the skin that range in color from white to black and
brown. The spots begin as small, rough bumps which thicken to look
like warts, moles, actinic keratoses, or skin cancer. Seborrheic
keratoses often have a waxy, "pasted-on-the-skin" look.
Seborrheic keratosis rarely causes any serious medical problems,
whereas actinic keratosis often progresses to skin cancer. These
two skin problems can often be differentiated based on the
appearance of the lesions, although a skin biopsy may be needed to
definitively diagnose the skin problems. Nonetheless, regardless of
possible outcomes, both skin conditions are cosmetically
undesirable for many people and furthermore can become inflamed and
uncomfortable.
Prescription medications do exist for the treatment of age spots
and other skin conditions related to hyperpigmentation. Some use
bleaching creams to fade the age spots gradually. These
prescription creams usually contain hydroquinone, with or without
retinoids, such as tretinoin. Bleaching creams usually take several
months to fade age spots. Other topical prescription creams, such
as Imiquimod, 5-Fluorouracil, and diclofenac-based Solaraze, work
by either directly attacking precancerous cells or indirectly
stimulating the body's immune system to recognize precancerous
cells. These creams, however, can produce a number of undesirable
side-effects including redness of skin, scabbing and crusting,
soreness, fever, achy joints and mouth sores.
Sometimes, procedural remedies can be utilized for age spot and/or
hyperpigmentation treatment. Examples of medical procedures for
treating actinic and seborrheic keratoses include laser treatment
to destroy cells that produce melanin; chemical peels to burn the
outer skin layer and allow new skin to grow in its place;
dermabrasion, which sands off the outer layers of the skin so new
skin can grow in its place; and cryosurgery, which freezes age
spots with liquid nitrogen. As with prescription drugs, each of
these medical procedures carries the risk of side effects and
complications.
There are also many over-the-counter and cosmetic preparations and
creams that are marketed for diminishing the appearance of
hyperpigmented skin and age spots. These creams are not as strong
as prescription creams, and the great majority may not effectively
remove excessive skin pigmentation. These creams mostly contain
hydroquinone, deoxyarbutin, glycolic acid, alpha hydroxy acid, or
kojic acid.
To avoid the potentially more invasive and extensive treatment
required if an age spot were to become malignant, preventing the
conversion of large sized, multiple, or inflamed actinic keratoses
into squamous cell carcinoma is important. Furthermore, from the
standpoint of skin appearance, many people will go to great costs
to cover up or try to diminish the appearance of both actinic and
seborrheic keratoses, as well as many other hyperpigmentation
conditions.
Another common skin condition is acne, or acne vulgaris, which
results from the action of hormones and other impurities at the
skin's oil glands and hair follicles. This condition affects about
85% of people to some degree in their lifetime. Acne is a
systematic inflammatory disease resulting from blockage of
sebaceous glands and hair follicles, which can become infected by
the bacterium Propionbacterium acnes (P. acnes). Sebum provides a
nutrient source for P. acnes, thus creating an ideal environment
for the bacteria to proliferate and cause the occurrence of
comedones, or "pimples."
Comedones are typically treated using creams or ointments
containing active drug materials, such as sebum secretion
inhibitors, keratinization inhibitors, antimicrobial agents and/or
anti-inflammatory agents; however, many of these drug materials
come with negative side effects. For example, mild inflammatory
acne is usually treated with common over the counter (OTC) topical
medications, including benzoyl peroxide, salicylic acid, or
retinoids. Benzoyl peroxide can leave the skin with an opaque or
white appearance, and can be extremely drying. Additionally,
benzoyl peroxide can bleach clothing and linens that contact the
treated area. Furthermore, some government regulations in various
parts of the world limit or exclude its use due to its potential
toxicity.
Alternatives to benzoyl peroxide are known, but are also associated
with certain negative side effects. For example, women can be
prescribed formulations containing estrogen and estrogen-like
compounds, such as ethinyl estradiol, which are often in the form
of contraceptive drugs. These drugs are typically effective as
sebum secretion inhibitors, but their use can be associated with
nausea, headaches, weight gain, mood swings, depression, and other
side-effects.
In addition, some acne treatments use an antimicrobial component
that includes chlorhexidine gluconate and benzalkonium chloride.
These antimicrobials can cause irritation and extreme chapping of
the skin. Furthermore antimicrobials can lead to resistant strains
developing in the wild-type fauna.
Comedolytic agents, such as salicylic acid, AHAs and the salts of
both acids, are popular for exfoliating dead skin cells and opening
and draining the pores. Comedolytic agents have limitations, and at
high concentrations with very acidic pH, they can cause significant
irritation. Comedolytic agents also have shown limited effect in
preventing proliferation of P. acnes via antimicrobial activity and
also have limited, if any, impact on directly reducing
inflammation.
Skin health is crucial for a long, healthy life. Additionally, skin
health is often an external expression of beauty and youthfulness.
There are a wide variety of products and treatment options for
treating and/or preventing a variety of skin conditions. Many of
these treatments, however, utilize harsh prescriptions or
procedures that have negative side-effects. Other, gentler options
are simply not effective for everyone. Thus, there is a need for
safe and effective cosmetic solutions that are capable of treating
and/or preventing a wide range of skin conditions, including, for
example, age spots and acne.
BRIEF SUMMARY OF THE INVENTION
The present invention provides microbe-based products, as well as
methods of their use, in topical cosmetic compositions. More
specifically, the present invention provides materials and methods
for treating certain conditions of the skin using a topical
cosmetic composition. Advantageously, the topical compositions and
methods of the subject invention are environmentally-friendly,
non-toxic, non-pharmaceutical, and cost-effective.
In certain embodiments, the skin condition is actinic keratosis, or
age spots. The subject invention provides materials and methods for
treating, preventing, removing and/or reducing the appearance of
age spots and other hyperpigmentation conditions of the skin, by
using biochemical-producing microbes and/or by-products of their
growth.
In certain embodiments, the skin condition is acne. The subject
invention provides materials and methods for treating, preventing,
removing and/or reducing the appearance of acne and other
blemish-causing conditions of the skin, by using
biochemical-producing microbes and/or by-products of their
growth.
In certain embodiments, the skin condition is another skin
condition, such as, for example, scars, body odor, aging-related
conditions (e.g., wrinkles, looseness and dryness), scalp
conditions (e.g., dandruff, seborrheic dermatitis and hair loss),
and in general, damage due to make-up, oil, pollution and other
impurities.
In preferred embodiments, the present invention utilizes
microorganisms and/or their growth by-products. For example,
embodiments of the present invention provide a topical composition
for treating a skin condition, wherein the composition comprises
biological amphiphilic molecules (e.g., biosurfactants) and/or
enzymes produced by microorganisms. In some embodiments, the
microbial growth by-products can have anti-microbial
properties.
In certain embodiments, the composition comprises a therapeutically
effective amount of biosurfactants selected from one or more
glycolipids, such as mannosylerythritol lipids (MELs),
sophorolipids (SLPs), rhamnolipids (RLPs) and trehalose lipids;
and/or one or more lipopeptides, such as surfactin, iturin and
fengycin. In certain embodiments, the biosurfactants according to
the present invention are capable of enhancing dermal penetration
of other active and inactive ingredients in the composition; thus
the biosurfactants can enhance the effectiveness of the treatment
while serving as active ingredients themselves.
In some embodiments, the topical composition further comprises
therapeutically effective amounts of resveratrol, hyaluronic acid
and/or anti-comedo agents, as well as other agents known to, for
example, heal, replenish, rejuvenate, moisturize, protect and/or
improve the appearance and/or health of the skin in any way (e.g.,
to reduce the appearance of scars).
In some embodiments, the topical composition can further comprise a
topically acceptable vehicle, such as a water-in-oil or
oil-in-water emulsion, or an aqueous serum.
In some embodiments, the topical cosmetic composition can further
comprise additional cosmetic adjuvants and additives typically
found in cosmetic compositions, such as, for example, organic
solvents, silicones, antimicrobials, stabilizers, thickeners,
softeners, sunscreens, moisturizers or fragrances. In one
embodiment, the topical composition further comprises a polymeric
stabilizer, such as, for example, poly(acrylic) acid.
The topical composition can be formulated as a suspension,
emulsion, hydrogel, multiphase solution, vesicular dispersion or
other known forms of topical cosmetics (e.g., a lotion, cream, gel
or ointment). Additionally, the topical composition can be
deliverable, e.g., in a squeeze tube or a cosmetic jar, as a pen, a
stick, a spray, in a wipe or via dropper.
In one embodiment, the subject invention provides methods of
treating a skin condition, wherein the topical cosmetic composition
is applied directly to an area of the skin where such a condition
exists. In some embodiments, "applying" the composition can
comprise leaving the composition on the skin, and/or rubbing it
into the skin to be absorbed therein. In some embodiments, the
composition can be applied to the skin for a therapeutically
effective amount of time and then rinsed from the skin using, for
example, water.
In certain embodiments, the topical cosmetic composition is
applied, e.g., every other day, once daily, or twice daily. In some
embodiments, the topical composition is applied every other day,
once daily, or twice daily, for an indefinite period of time, e.g.,
for at least one, two, three weeks, or longer, in order to achieve
and/or maintain the treatment of the skin condition.
The topical cosmetic compositions and methods of the subject
invention can be used to treat and/or prevent a variety of skin
conditions, including, for example, age spots, acne, scars, body
odor, aging-related conditions (e.g., wrinkles, looseness and
dryness), and/or scalp conditions (e.g., dandruff, seborrheic
dermatitis and hair loss). Additionally, the topical cosmetic
compositions can be used as a cleanser to remove makeup and other
impurities from the face and skin.
In certain embodiments, the subject invention provides
microbe-based compositions comprising cultivated microorganisms
and/or their growth by-products. Methods of producing the
microorganisms and their growth by-products are also provided.
In some embodiments, the microorganisms are biosurfactant-producing
bacteria (e.g., Bacillus subtilis, Bacillus amyloliquefaciens,
Rhodococcus erythropolis, Pseudomonas aeruginosa) or yeasts (e.g.,
Pseudozyma aphidis, Pichia spp., or Starmerella bombicola). In one
embodiment, the microorganisms are mutants of a desired strain or
species.
In one embodiment, the subject invention provides methods of
producing a biosurfactant, enzyme and/or other protein by
cultivating a microbe strain of the subject invention under
conditions appropriate for growth and biosurfactant, enzyme and/or
protein production; and purifying the biosurfactant, enzyme and/or
other protein. The cultivation process can be, for example,
submerged cultivation, solid state fermentation (SSF), and/or a
combination thereof.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1B show examples of the appearance of age spots on the
forehead and scalp of a subject.
FIG. 2 shows an example of the appearance of squamous cell
carcinoma on skin.
FIG. 3 shows an example of the appearance of seborrheic keratosis
on skin.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides microbe-based products, as well as
methods of their use, in topical cosmetic compositions. More
specifically, the present invention provides materials and methods
for treating certain conditions of the skin, such as, for example,
age spots and/or acne, using a topical cosmetic composition
comprising microbial growth by-products.
The topical cosmetic compositions and methods of the subject
invention can also be used to treat and/or prevent a variety of
other skin conditions, including, for example, scars; body odor;
aging-related conditions (e.g., wrinkles, looseness and dryness);
and scalp issues (e.g., dandruff, seborrheic dermatitis and hair
loss. Additionally, the topical cosmetic compositions can be used
as a cleanser to remove makeup and other impurities (e.g., dirt and
pollution) from the face and skin.
Advantageously, the topical compositions and methods of the subject
invention are environmentally-friendly, non-toxic,
non-pharmaceutical, and cost-effective.
Selected Definitions
As used herein, the term "skin condition" encompasses human and
animal conditions, disorders, or diseases affecting skin. Such skin
conditions include, but are not limited to, conditions involving
the epidermis, dermis (including connective tissue, sebaceous
glands and hair follicles), and the subcutaneous tissue
(hypodermis). Symptoms of skin conditions can include, for example,
acneiform symptoms, pigmentation or loss thereof, flushing,
inflammation, wrinkles, dryness, looseness, thickening, scaling,
scarring, flaking, rash, hives, blisters, ulcers, peeling, hair
loss and other changes in the appearance of the skin. Skin
conditions that can, in certain embodiments, be treated and/or
preventing using compositions, products and methods described
herein include, but are not limited to, acne, blemishes, rosacea,
folliculitis, carcinoma, melanoma, perioral dermatitis, cellulitis,
carbuncles, photodamage, skin aging (e.g., wrinkles and dryness),
age spots, scars, lupus, psoriasis, ichtiosis, atopic dermatitis,
chronic wounds, bed sores, keratosis piralis, sebaceous cysts,
vitiligo, melisma, warts, inflammatory dermatoses, post
inflammatory hyperpigmentation, keratoses, eczema, xerosis,
pruritis, lichen planus, nodular prurigo, microbial infection, body
odor, scalp conditions and miliaria.
As used herein, the term "subject" refers to an animal, especially
a mammal, receiving medical treatment, including over-the-counter
medical treatment and preventative care. The preferred subject in
the context of this invention is a human patient. The subject can
be of any age or stage of development including baby, infant,
toddler, preteen, teenager, and adult. The subject can be any
gender.
As used herein, "cosmetically acceptable," "topically acceptable"
and "dermatologically acceptable" are used interchangeably and are
intended to mean that a particular component is safe and non-toxic
for application to a human integument (e.g., skin) at the levels
employed. In one embodiment, the components of the composition are
recognized as being Generally Regarded as Safe (GRAS).
As used herein, the terms "therapeutically effective amount,"
"effective amount," and "effective dose" are used to refer to an
amount of something (e.g., a compound, a composition, time) is
capable of treating a condition or disorder in a subject. The
actual amount will vary depending on a number of factors including,
but not limited to, the particular condition or disorder being
treated, the severity of the condition, the size, age, and health
of the subject, and the route of administration.
As used herein, the term "treatment" refers to eradicating,
reducing, ameliorating, or reversing, a degree, sign or symptom of
a condition or disorder to any extent, and includes, but does not
require, a complete cure of the condition or disorder. Treating can
be curing, improving, or partially ameliorating a disorder.
As used herein, "preventing" a condition or disorder refers to
avoiding, delaying, forestalling, or minimizing the onset of a
particular sign or symptom of the condition or disorder. Prevention
can, but is not required to be, absolute or complete, meaning the
sign or symptom may still develop at a later time. Prevention can
include reducing the severity of the onset of such a condition or
disorder, and/or inhibiting the progression of the condition or
disorder to a more severe condition or disorder. For example, in
one embodiment, preventing hyperpigmentation can refer to avoiding,
delaying, forestalling, or minimizing one or more unwanted features
associated with skin hyperpigmentation, such as reducing the
darkness or size of hyperpigmented areas that eventually develop.
As another example, in one embodiment, preventing acne can refer to
avoiding, delaying, forestalling, or minimizing one or more
unwanted features associated with acne, such as reducing the
number, darkness, and/or size of comedones that eventually develop,
lessening the severity of acne that eventually develops, and/or
completely or almost completely preventing the growth of P. acnes,
the development of acne blemishes, and the other symptoms of
acne.
As used herein, a "microbe-based composition" means a composition
that comprises components that were produced as the result of the
growth of microorganisms or other cell cultures. Thus, the
microbe-based composition may comprise the microbes themselves
and/or by-products of microbial growth (e.g., biosurfactants,
solvents and/or enzymes). The cells may be in a vegetative state or
in spore form, or a mixture of both. The cells may be planktonic or
in a biofilm form, or a mixture of both. The cells may be intact or
lysed. The cells can be present, with broth in which they were
grown, at, for example, a concentration of 1.times.10.sup.4,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10, or
1.times.10.sup.11 or more cells per milliliter of the composition.
In one embodiment, the microbe-based composition may comprise only
the broth in which the cells were grown, with the cells removed.
The by-products of growth may be present in the broth and can
include, for example, metabolites, cell membrane components,
expressed proteins, and/or other cellular components.
The subject invention further provides "microbe-based products,"
which are products that are to be applied in practice to achieve a
desired result. The microbe-based product can be simply the
microbe-based composition harvested from the microbe cultivation
process. Alternatively, the microbe-based product may comprise
further ingredients that have been added. These additional
ingredients can include, for example, stabilizers, buffers,
appropriate carriers, such as water, salt solutions, or any other
appropriate carrier, added nutrients to support further microbial
growth, non-nutrient growth enhancers, and/or agents that
facilitate tracking of the microbes and/or the composition in the
environment to which it is applied. The microbe-based product may
also comprise mixtures of microbe-based compositions. The
microbe-based product may also comprise one or more components of a
microbe-based composition that have been processed in some way such
as, but not limited to, filtering, centrifugation, lysing, drying,
purification and the like.
A "metabolite" refers to any substance produced by metabolism or a
substance necessary for taking part in a particular metabolic
process. A metabolite can be an organic compound that is a starting
material (e.g., glucose), an intermediate (e.g., acetyl-CoA) in, or
an end product (e.g., n-butanol) of metabolism. Examples of
metabolites can include, but are not limited to, enzymes, toxins,
acids, solvents, alcohols, proteins, carbohydrates, vitamins,
minerals, microelements, amino acids, polymers, and
surfactants.
As used herein, the terms "isolated" or "purified," when used in
connection with biological or natural materials such as nucleic
acid molecules, polynucleotides, polypeptides, proteins, organic
compounds, such as small molecules, microorganism cells/strains, or
host cells, means the material is substantially free of other
compounds, such as cellular material, with which it is associated
in nature. That is, the materials do not occur naturally without
these other compounds and/or have different or distinctive
characteristics compared with those found in the native
material.
In certain embodiments, purified compounds are at least 60% by
weight the compound of interest. Preferably, the preparation is at
least 75%, more preferably at least 90%, and most preferably at
least 99% or 100% (w/w) of the desired compound by weight. Purity
is measured by any appropriate standard method, for example, by
column chromatography, thin layer chromatography, or
high-performance liquid chromatography (HPLC) analysis.
As used herein, "surfactant" means a compound that lowers the
surface tension (or interfacial tension) between two liquids or
between a liquid and a solid. Surfactants act as detergents,
wetting agents, emulsifiers, foaming agents, and/or dispersants. By
"biosurfactant" is meant a surface-active substance produced by a
living cell.
The transitional term "comprising," which is synonymous with
"including," or "containing," is inclusive or open-ended and does
not exclude additional, unrecited elements or method steps. By
contrast, the transitional phrase "consisting of" excludes any
element, step, or ingredient not specified in the claim. The
transitional phrase "consisting essentially of" limits the scope of
a claim to the specified materials or steps "and those that do not
materially affect the basic and novel characteristic(s)" of the
claimed invention.
Unless specifically stated or obvious from context, as used herein,
the term "or" is understood to be inclusive. Unless specifically
stated or obvious from context, as used herein, the terms "a," "an"
and "the" are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein,
the term "about" is understood as within a range of normal
tolerance in the art, for example, within 2 standard deviations of
the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value.
The recitation of a listing of chemical groups in any definition of
a variable herein includes definitions of that variable as any
single group or combination of listed groups. The recitation of an
embodiment for a variable or aspect herein includes that embodiment
as any single embodiment or in combination with any other
embodiments or portions thereof.
Any compositions or methods provided herein can be combined with
one or more of any of the other compositions and methods provided
herein.
Other features and advantages of the invention will be apparent
from the following description of the preferred embodiments
thereof, and from the claims. All references cited herein are
hereby incorporated by reference.
Topical Cosmetic Compositions
The subject invention provides skin care compositions and methods
of their use. In particular, the subject invention provides skin
care and cosmetic products that can treat and/or prevent a skin
condition, including age spots, acne, scars, body odor,
aging-related conditions, scalp conditions, and/or others described
herein.
In certain embodiments, the present invention utilizes microbial
growth by-products. Particularly, embodiments of the present
invention provide a topical composition for treating human skin
conditions, wherein the composition comprises biological
amphiphilic molecules (e.g., biosurfactants) produced by the
cultivation of biochemical-producing microorganisms. In some
embodiments, the biological amphiphilic molecules are utilized in a
crude form, wherein the molecule is present in the broth in which
the microorganism is cultivated and is collected therefrom without
purification. The crude form can comprise, for example, at least
20%, 30%, 40%, 50%, 60%, 70% or 80% amphiphilic molecule in broth.
In some embodiments, the biological amphiphilic molecules have been
purified from the products of cultivation.
In certain embodiments, the composition can comprise a
therapeutically effective amount of glycolipids, such as
mannosylerythritol lipids (MELs), sophorolipids (SLPs), trehalose
lipids (TLs) and rhamnolipids (RLP); and/or lipopeptides, such as
surfactin, iturin A, and fengycin. In one embodiment, the
composition can comprise a combination of any of these
biosurfactants.
The biological amphiphilic molecules according to the present
invention are capable of one or more of the following: killing
pathogenic agents in the skin, modulating the skin's immune system,
killing melanocytes to allow for replacement cells to grow,
reducing oxidative stress, enhancing multiplication and function of
keratinocytes and fibroblasts, and enhancing dermal penetration of
both the, e.g., biosurfactants, and one or more other active
ingredients in the composition. Thus, while providing therapeutic
benefits themselves, these beneficial molecules can also enhance
the overall effectiveness of the topical composition in treating
skin conditions related to, for example, hyperpigmentation or the
presence of microbial agents.
MELs and SLPs are part of a glycolipid class of biosurfactants
produced by a variety yeasts. MELs are produced mainly by the yeast
genus Pseudozyma, with significant variability among MEL structures
produced by each species. MELs are non-toxic and are stable at wide
temperatures and pH ranges. Furthermore, MELs can be used without
any additional preservatives.
In preferred embodiments, MEL concentration in the topical cosmetic
composition ranges from 0.001% to 90% of the total composition by
weight, from 0.01% to 50%, from 0.05% to 10%, and preferably from
0.1% to 2.0%.
SLPs are produced in large quantity by several nonpathogenic yeast
species, the most studied of which is Starmerella bombicola. SLPs
have environmental compatibility, high biodegradability, low
toxicity, high selectivity and specific activity in a broad range
of temperature, pH and salinity conditions.
In preferred embodiments, SLP concentration in the topical cosmetic
composition ranges from 0.001% to 90% of the total composition by
weight, from 0.01% to 50%, from 0.05% to 10%, and preferably from
0.1% to 2.0%. In one embodiment, the topical composition comprises
SLP in acidic form.
RLPs are glycolipids produced mainly by Pseudomonas bacteria. They
are natural emulsifiers, and can be used according to the subject
invention to replace non-biological surfactants, such as sodium
lauryl sulfate, sodium dodecyl sulfate and sodium laureth sulfate,
in a cosmetic composition. Furthermore, RLPs can be formulated to
increase moisture retention or to lubricate skin, minimize the
appearance of wrinkles, and increase smoothness of skin. Even
further, RLPs can be used as antibacterial (Gram-positive) and
antifungal agents.
In preferred embodiments, RLP concentration in the topical cosmetic
composition ranges from 0.001% to 90% of the total composition by
weight, from 0.01% to 50%, from 0.05% to 10%, and preferably from
0.1% to 2.0%.
Trehalose lipids (TLs) are glycolipids produced by, for example,
the bacteria Rhodococcus erythropolis. TLs possess emulsifying and
dispersing characteristics. They exhibit increased levels of
surface activity and have certain antiviral and antimicrobial
properties.
In preferred embodiments, TL concentration in the topical cosmetic
composition ranges from 0.001% to 90% of the total composition by
weight, from 0.01% to 50%, from 0.05% to 10%, and preferably from
0.1% to 2.0%.
Surfactin is a lipopeptide produced by certain bacterial strains,
mainly Bacillus subtilis. Surfactin has high level surface
activating function, and is extremely hydrophilic, forming a
transparent gel at a wider range of concentrations than other
biosurfactants. This biosurfactant can act as a skin penetration
agent for cosmetic products, a foaming agent and an emulsifier.
Furthermore, surfactin exhibits effective antibacterial
(Gram-negative), antifungal and antiviral properties.
In preferred embodiments, surfactin concentration in the topical
cosmetic composition ranges from 0.001% to 90% of the total
composition by weight, from 0.01% to 50%, from 0.05% to 10%, from
0.1% to 5.0%, and preferably from 0.01% to 2.0%.
Additional biological amphiphilic molecules useful according to the
present invention include mannoprotein, beta-glucan and other
metabolites that have bio-emulsifying and surface/interfacial
tension-reducing properties.
In some embodiments, the topical cosmetic composition can comprise
therapeutically effective amounts of enzymes and/or proteins
produced by microorganisms. For example, from about 0.001% to about
20% by weight, preferably from about 0.01% to about 15% by weight,
or from about 0.05% to about 10% by weight, of one or more enzymes
and/or proteins can be included. These can include, but are not
limited to, exo-beta-1,3-glucanase, "killer toxins," chitinase,
esterases, lipases, glycosidases, amylases, and proteases
beneficial for improving skin health.
In some embodiments, the topical composition further comprises
therapeutically effective amounts of resveratrol. In certain
embodiments, amount of resveratrol with respect to total weight of
the subject topical composition ranges from 0.001 to 5.0% by
weight, more preferably from 0.05 to 2.0% by weight, and most
preferably from 0.2 to 1.0% by weight.
Resveratrol is a naturally-occurring substance found in the skin of
fruits such as grapes, blueberries, raspberries and mulberries. It
is reported to be an extremely potent antioxidant, a modulator of
genetic expression via signal transduction, an inhibitor of
inflammatory mediators and, by acting on diverse mechanisms
simultaneously, it has been emphasized as a promising,
multi-target, anticancer agent, relevant in both cancer prevention
and treatment. Additionally, resveratrol has unique skin bleaching
abilities, as it reduces the synthesis of melanin.
In one embodiment, the topical composition can further comprise a
polymeric stabilizer, such as, for example, from about 0.01% to
about 5.0%, or from about 0.05% to about 2.0%, or from about 0.5%
to about 1.0% poly(acrylic) acid. Poly(acrylic) acid helps to
prevent resveratrol from crystallizing.
In some embodiments, the topical composition further comprises
therapeutically effective amounts of hyaluronic acid. Hyaluronic
acid is produced naturally in the fibroblasts of human skin, and
can be used in the healing of skin wounds such as burns and ulcers,
and as a skin moisturizer. Hyaluronic acid can aide in moisture
retention, tissue repair, and holding together the collagen and
elastin that make up the structural components of skin. It can also
help create a protective barrier against undesirable
microorganisms.
In one embodiment, the topical composition comprises from about
0.01% to about 10.0%, or from about 0.05% to about 8.0%, from about
0.5% to about 5.0%, or from about 1.0% to about 3.0% by weight
hyaluronic acid.
In some embodiments, the topical composition can further comprise a
topically or cosmetically acceptable vehicle.
The cosmetically acceptable vehicle may be in the form of an
emulsion. Non-limiting examples of suitable emulsions include
water-in-oil emulsions, oil-in-water emulsions, silicone-in-water
emulsions, water-in-silicone emulsions, wax-in-water emulsions,
water-oil-water triple emulsions or the like having the appearance
of a cream, gel or microemulsions.
As used herein, the term "oil" includes silicone oils unless
otherwise indicated. The emulsion may include an emulsifier, such
as a nonionic, anionic or amphoteric surfactant, or a gallant,
typically in an amount from about 0.001% to about 5% by weight.
The cosmetically acceptable vehicle may include water; vegetable
oils; mineral oils; ester oils such as octal palmitate, isopropyl
myristate and isopropyl palmitate; ethers such as dicapryl ether
and dimethyl isosorbide; alcohols such as ethanol and isopropanol;
fatty alcohols such as cetyl alcohol, cetearyl alcohol, stearyl
alcohol and behenyl alcohol; isoparaffins such as isooctane,
isododecane (IDD) and isohexadecane; silicone oils such as
cyclomethicone, dimethicone, dimethicone cross-polymer,
polysiloxanes and their derivatives, preferably organomodified
derivatives including PDMS, dimethicone copolyol, dimethiconols,
and amodimethiconols; hydrocarbon oils such as mineral oil,
petrolatum, isoeicosane and polyolefins, e.g., (hydrogenated)
polyisobutene; polyols such as propylene glycol, glycerin, butylene
glycol, pentylene glycol, hexylene glycol, caprylyl glycol; waxes
such as beeswax, carnauba, ozokerite, microcrystalline wax,
polyethylene wax, and botanical waxes; or any combinations or
mixtures of the foregoing. Aqueous vehicles may include one or more
solvents miscible with water, including lower alcohols, such as
ethanol, isopropanol, and the like. The vehicle may comprise from
about 1% to about 99% by weight of the composition, from 10% to
about 85%, from 25% to 75%, or from 50% to about 65%.
In some embodiments, the topical cosmetic composition can further
comprise additional cosmetic adjuvants and additives commonly
included in cosmetic compositions, such as, for example, organic
solvents, stabilizers, silicones, thickeners, softeners,
sunscreens, moisturizers or fragrances. The amounts of each
ingredient, whether active or inactive, are those conventionally
used in the cosmetic field to achieve their intended purpose, and
typically range from about 0.0001% to about 25%, or from about
0.001% to about 20% of the composition, although the amounts may
fall outside of these ranges. The nature of these ingredients and
their amounts must be compatible with the production and function
of the compositions of the disclosure.
In one embodiment, the composition may include additional skin
actives, including but not limited to, retinoids, botanicals,
keratolytic agents, desquamating agents, keratinocyte proliferation
enhancers, collagenase inhibitors, elastase inhibitors,
depigmenting agents, anti-inflammatory agents, steroids, anti-acne
agents, antioxidants, and advanced glycation end-product (AGE)
inhibitors, to name but a few.
In one embodiment, the composition may include additional
anti-aging components, including, but not limited to, botanicals
(e.g., Butea frondosa extract, Aloe vera extract); phytol; phytonic
acid; phospholipids; silicones; petrolatum; triglycerides; omerga
fatty acids; retinoids; hydroxy acids (including alpha-hydroxy
acids and beta-hydroxy acids), salicylic acid and alkyl
salicylates; exfoliating agents (e.g., glycolic acid,
3,6,9-trioxaundecanedioic acid, etc.), estrogen synthetase
stimulating compounds (e.g., caffeine and derivatives); compounds
capable of inhibiting 5 alpha-reductase activity (e.g., linolenic
acid, linoleic acid, finasteride, and mixtures thereof); and
barrier function enhancing agents (e.g., ceramides, glycerides,
cholesterol and its esters, alpha-hydroxy and omega-hydroxy fatty
acids and esters thereof.)
Exemplary retinoids include, without limitation, retinoic acid
(e.g., all-trans, or 9-cis, or 13-cis), and derivatives thereof,
retinaldehyde, retinol (Vitamin A) and esters thereof, such as
retinyl palmitate, retinyl acetate and retinyl propionate, and
salts thereof. When present, the retinoids will typically be
included in amounts from about 0.0001% to about 5% by weight, more
typically from about 0.01% to about 2.5% by weight, or from about
0.1% to about 1.0% by weight. Compositions according to this
embodiment will typically include an antioxidant such as ascorbic
acid and/or BHT and/or a chelating agent such as EDTA or a salt
thereof (e.g., disodium EDTA).
In another embodiment, the topical compositions of the present
invention may also include one or more of the following: a skin
penetration enhancer; an emollient, such as isopropyl myristate,
petrolatum, volatile or non-volatile silicones oils (e.g.,
methicone, dimethicone), ester oils, mineral oils, and fatty acid
esters; a humectant, such as glycerin, hexylene glycol or caprylyl
glycol; a skin plumper, such as palmitoyl oligopeptide, collagen,
collagen and/or glycosaminoglycan (GAG) enhancing agents; a
sunscreen, such as avobenzone or octyl methoxycinnamate; an
exfoliating agent; and an antioxidant.
Suitable exfoliating agents include, for example, alpha-hydroxy
acids, beta-hydroxy acids, oxa-acids, oxadiacids, and their
derivatives such as esters, anhydrides and salts thereof. Suitable
hydroxy acids include, for example, glycolic acid, lactic acid,
malic acid, tartaric acid, citric acid, 2-hydroxyalkanoic acid,
mandelic acid, salicylic acid and derivatives thereof. One
exemplary exfoliating agent is glycolic acid. When present, the
exfoliating agent may comprise from about 0.001% to about 20% by
weight of the composition.
Examples of antioxidants that may be used in the present
compositions include compounds having phenolic hydroxy functions,
such as ascorbic acid and its derivatives/esters; beta-carotene;
catechins; curcumin; ferulic acid derivatives (e.g., ethyl
ferulate, sodium ferulate); gallic acid derivatives (e.g., propyl
gallate); lycopene; reductic acid; rosmarinic acid; tannic acid;
tetrahydrocurcumin; tocopherol and its derivatives, including
tocopheryl acetate; uric acid; or any mixtures thereof. Other
suitable antioxidants are those that have one or more thiol
functions (--SH), in either reduced or non-reduced form, such as
glutathione, lipoic acid, thioglycolic acid, and other sulfhydryl
compounds. The antioxidant may be inorganic, such as bisulfites,
metabisulfites, sulfites, or other inorganic salts and acids
containing sulfur. Antioxidants may comprise, individually or
collectively, from about 0.001% to about 10% (w/w), or from about
0.01% to about 5% (w/w) of the total weight of the composition.
Non-biological surfactants can also be added to the formulation.
Examples of surfactants include, but are not limited to, alkyl
sulfates, alkyl ether sulfates (e.g., sodium/ammonium lauryl
sulfates and sodium/ammonium laureth sulfates), amphoterics (e.g.,
amphoacetates and amphopropionates), sulfosuccinates, alkyl
polyglucosides, betaines (e.g., cocamidopropul betaine (CAPB)),
sultaines, sacrosinates, isethionates, taurates, ethoxylated
sorbitan esters, alkanolamides and amino-acid based
surfactants.
Viscosity modifiers can also be added to the compositions,
including, for example, cocamide DEA, oleamide DEA, sodium
chloride, cellulosic polymers, polyacrylates, ethoxylated esters,
alcohol, glycols, xylene sulfonates, polysorbate 20, alkanolamides,
and cellulose derivatives (e.g., hydroxypropyl methylcellulose and
hydroxyethyl cellulose).
Polymers can also be added, include, for example, xanthan gum guar
gum, polyquaternium-10, PEG-120 methyl glucose dioleate, PEG-150
distearate, PEG-150 polyglyceryl-2 tristearate and PEG-150
pentaerythrityl tetrastearate
Other additives include: vitamins, such as tocopherol and ascorbic
acid; vitamin derivatives such as ascorbyl monopalmitate,
tocopheryl acetate, and Vitamin E palmitate; thickeners such as
hydroxyalkyl cellulose, carboxymethylcellulose, carbomers, and
vegetable gums such as xanthan gum; gelling agents, such as
ester-terminated polyester amides; structuring agents; metal
chelating agents such as EDTA or salts thereof; pigments;
colorants; proteins, such as lactoferrin; and pH adjusters (citric
acid, ethanolamine, sodium hydroxide, etc.).
The composition may optionally comprise other components known to
those skilled in the art including, but not limited to, film
formers, moisturizers, minerals, viscosity and/or rheology
modifiers, anti-acne agents, insect repellents, skin cooling
compounds, skin protectants, lubricants, fragrances, preservatives,
stabilizers, and mixtures thereof. In addition to the foregoing,
the cosmetic compositions of the invention may contain any other
compound for the treatment of skin conditions or disorders.
In addition, the compositions contemplated by this disclosure can
include one or more compatible cosmetically acceptable adjuvants
commonly used and known by the skilled practitioner, such as
colorants, pearls, chromalites, micas, pigments, dyes, fragrances,
emollients, humectants, preservatives, conditioners, vitamins,
chelators, thickeners, anesthetics, anti-allergenics, antifungals,
antimicrobials, other anti-inflammatory agents, antioxidants,
antiseptics, depigmenting agents, film formers, insect repellents,
pharmaceutical agents, photostabilizing agents, sunscreens,
stabilizers, surfactants, thickeners, viscosity modifiers, and
botanicals. The topical compositions of the present disclosure may
also include a skin penetration enhancer, a surface smoother, a
skin plumper, an optical diffuser, an exfoliation promoter, and an
antioxidant. Details with respect to these and other suitable
cosmetic ingredients can be found in the "International Cosmetic
Ingredient Dictionary and Handbook," 10th Edition (2004), published
by the Cosmetic, Toiletry, and Fragrance Association (CTFA), at pp.
2177-2299, which is herein incorporated by reference in its
entirety. The amounts of these various substances are those that
are conventionally used in the cosmetic or pharmaceutical fields,
for example, they can constitute from about 0.01% to about 20% of
the total weight of the composition.
A sunscreen or combination of sunscreens may be included to protect
the skin from both UVA and UVB rays. Among the sunscreens that can
be employed in the present compositions are avobenzone, cinnamic
acid derivatives (such as octylmethoxy cinnamate), octyl
salicylate, oxybenzone, octocrylene, titanium dioxide, zinc oxide,
or any mixtures thereof. The sunscreen may be present from about 1
wt % to about 30 wt % of the total weight of the composition.
Additionally, the composition may contain compounds capable of
bleaching keratoses dark and reddish colors and the compounds that
can improve the skin turgor and increase the penetration of other
compounds into the deeper layers of the skin.
The composition may be formulated as a suspension, emulsion,
hydrogel, multiphase solution, vesicular dispersion or in any other
known form of cosmetic topical skin composition.
In certain embodiments, the topical cosmetic composition may be
formulated so that it can be applied, for example, via pen, tube,
bottle, brush, stick, sponge, cotton swab, towelette (wipe),
sprayer, dropper, hand or finger.
The composition may be formulated in a variety of product forms,
such as, for example, a lotion, cream, serum, spray, aerosol,
liquid cake, ointment, essence, gel, paste, patch, pencil, powder,
towelette, soap, shampoo, conditioner, stick, foam, mousse, elixir
or concentrate. In preferred embodiments, the composition is
formulated so that is particularly suitable for topical
administration to the skin.
The composition can be formulated within a wide range of pH levels.
In one embodiment, the pH of the topical composition ranges from
1.0 to 13.0. In some embodiments, the pH of the topical composition
ranges from 2.0 to 12.0. Other pH ranges suitable for the subject
composition include from 3.5 to 7.0, or from 7.0 to 10.5. Suitable
pH adjusters such as sodium hydroxide, citric acid and
triethanolamine may be added to bring the pH within the desired
range.
Growth of Microbes and Production of Microbial Growth
By-Products
The subject invention provides methods for cultivating
microorganisms and production of microbial metabolites and/or other
by-products of microbial growth. The microbial cultivation systems
would typically use submerged culture fermentation; however,
surface culture and hybrid systems can also be used. As used herein
"fermentation" refers to growth of cells under controlled
conditions. The growth could be aerobic or anaerobic.
In one embodiment, the subject invention provides materials and
methods for the production of biomass (e.g., viable cellular
material), extracellular metabolites (e.g. small molecules and
excreted proteins), residual nutrients and/or intracellular
components (e.g. enzymes and other proteins).
The microbe growth vessel used according to the subject invention
can be any fermenter or cultivation reactor for industrial use. In
one embodiment, the vessel may have functional controls/sensors or
may be connected to functional controls/sensors to measure
important factors in the cultivation process, such as pH, oxygen,
pressure, temperature, agitator shaft power, humidity, viscosity
and/or microbial density and/or metabolite concentration.
In a further embodiment, the vessel may also be able to monitor the
growth of microorganisms inside the vessel (e.g., measurement of
cell number and growth phases). Alternatively, a daily sample may
be taken from the vessel and subjected to enumeration by techniques
known in the art, such as dilution plating technique. Dilution
plating is a simple technique used to estimate the number of
microbes in a sample. The technique can also provide an index by
which different environments or treatments can be compared.
In one embodiment, the method includes supplementing the
cultivation with a nitrogen source. The nitrogen source can be, for
example, potassium nitrate, ammonium nitrate ammonium sulfate,
ammonium phosphate, ammonia, urea, and/or ammonium chloride. These
nitrogen sources may be used independently or in a combination of
two or more.
The method can provide oxygenation to the growing culture. One
embodiment utilizes slow motion of air to remove low-oxygen
containing air and introduce oxygenated air. The oxygenated air may
be ambient air supplemented daily through mechanisms including
impellers for mechanical agitation of the liquid, and air spargers
for supplying bubbles of gas to the liquid for dissolution of
oxygen into the liquid.
The method can further comprise supplementing the cultivation with
a carbon source. The carbon source is typically a carbohydrate,
such as glucose, sucrose, lactose, fructose, trehalose, mannose,
mannitol, and/or maltose; organic acids such as acetic acid,
fumaric acid, citric acid, propionic acid, malic acid, malonic
acid, and/or pyruvic acid; alcohols such as ethanol, propanol,
butanol, pentanol, hexanol, isobutanol, and/or glycerol; fats and
oils such as soybean oil, rice bran oil, olive oil, corn oil,
sesame oil, and/or linseed oil; etc. These carbon sources may be
used independently or in a combination of two or more.
In one embodiment, growth factors and trace nutrients for
microorganisms are included in the medium. This is particularly
preferred when growing microbes that are incapable of producing all
of the vitamins they require. Inorganic nutrients, including trace
elements such as iron, zinc, copper, manganese, molybdenum and/or
cobalt may also be included in the medium. Furthermore, sources of
vitamins, essential amino acids, and microelements can be included,
for example, in the form of flours or meals, such as corn flour, or
in the form of extracts, such as yeast extract, potato extract,
beef extract, soybean extract, banana peel extract, and the like,
or in purified forms. Amino acids such as, for example, those
useful for biosynthesis of proteins, can also be included, e.g.,
L-Alanine.
In one embodiment, inorganic salts may also be included. Usable
inorganic salts can be potassium dihydrogen phosphate, dipotassium
hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate,
magnesium chloride, iron sulfate, iron chloride, manganese sulfate,
manganese chloride, zinc sulfate, lead chloride, copper sulfate,
calcium chloride, calcium carbonate, and/or sodium carbonate. These
inorganic salts may be used independently or in a combination of
two or more.
In some embodiments, the method for cultivation may further
comprise adding additional acids and/or antimicrobials in the
liquid medium before and/or during the cultivation process.
Antimicrobial agents or antibiotics are used for protecting the
culture against contamination. Additionally, antifoaming agents may
also be added to prevent the formation and/or accumulation of foam
when gas is produced during cultivation.
The pH of the mixture should be suitable for the microorganism of
interest. Buffers, and pH regulators, such as carbonates and
phosphates, may be used to stabilize pH near a preferred value.
When metal ions are present in high concentrations, use of a
chelating agent in the liquid medium may be necessary.
The method and equipment for cultivation of microorganisms and
production of the microbial by-products can be performed in a
batch, quasi-continuous, or continuous processes.
In one embodiment, the method for cultivation of microorganisms is
carried out at about 5.degree. to about 100.degree. C., preferably,
15 to 60.degree. C., more preferably, 25 to 50.degree. C. In a
further embodiment, the cultivation may be carried out continuously
at a constant temperature. In another embodiment, the cultivation
may be subject to changing temperatures.
In one embodiment, the equipment used in the method and cultivation
process is sterile. The cultivation equipment such as the
reactor/vessel may be separated from, but connected to, a
sterilizing unit, e.g., an autoclave. The cultivation equipment may
also have a sterilizing unit that sterilizes in situ before
starting the inoculation. Air can be sterilized by methods know in
the art. For example, the ambient air can pass through at least one
filter before being introduced into the vessel. In other
embodiments, the medium may be pasteurized or, optionally, no heat
at all added, where the use of low water activity and low pH may be
exploited to control bacterial growth.
In one embodiment, the subject invention provides methods of
producing a biosurfactant, enzyme and/or other protein by
cultivating a microbe strain of the subject invention under
conditions appropriate for growth and biosurfactant, enzyme and/or
protein production; and purifying the biosurfactant, enzyme and/or
other protein.
In one embodiment, the subject invention further provides a method
for producing other microbial metabolites such as ethanol, lactic
acid, beta-glucan, proteins, peptides, metabolic intermediates,
polyunsaturated fatty acid, and lipids. The metabolite content
produced by the method can be, for example, at least 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90%.
The biomass content of the fermentation broth may be, for example
from 5 g/l to 180 g/l or more. In one embodiment, the solids
content of the broth is from 10 g/l to 150 g/l.
The microbial growth by-product produced by microorganisms of
interest may be retained in the microorganisms or secreted into the
liquid medium. In another embodiment, the method for producing
microbial growth by-product may further comprise steps of
concentrating and purifying the microbial growth by-product of
interest. In a further embodiment, the liquid medium may contain
compounds that stabilize the activity of microbial growth
by-product.
In one embodiment, all of the microbial cultivation composition is
removed upon the completion of the cultivation (e.g., upon, for
example, achieving a desired cell density, or density of a
specified metabolite in the broth). In this batch procedure, an
entirely new batch is initiated upon harvesting of the first
batch.
In another embodiment, only a portion of the fermentation product
is removed at any one time. In this embodiment, biomass with viable
cells remains in the vessel as an inoculant for a new cultivation
batch. The composition that is removed can be a cell-free broth or
contain cells. In this manner, a quasi-continuous system is
created.
Advantageously, the method does not require complicated equipment
or high energy consumption. The microorganisms of interest can be
cultivated at small or large scale on site and utilized, even being
still-mixed with their media. Similarly, the microbial metabolites
can also be produced at large quantities at the site of need.
The microorganisms grown according to the systems and methods of
the subject invention can be, for example, bacteria, yeast and/or
fungi. These microorganisms may be natural, or genetically modified
microorganisms. For example, the microorganisms may be transformed
with specific genes to exhibit specific characteristics. The
microorganisms may also be mutants of a desired strain. As used
herein, "mutant" means a strain, genetic variant or subtype of a
reference microorganism, wherein the mutant has one or more genetic
variations (e.g., a point mutation, missense mutation, nonsense
mutation, deletion, duplication, frameshift mutation or repeat
expansion) as compared to the reference microorganism. Procedures
for making mutants are well known in the microbiological art. For
example, UV mutagenesis and nitrosoguanidine are used extensively
toward this end.
In preferred embodiments, the microbes are biosurfactant-producing
and/or enzyme-producing microorganisms. In one embodiment, the
microorganism is a yeast or fungus. Yeast and fungus species
suitable for use according to the current invention, include, for
example, Candida, Saccharomyces (S. cerevisiae, S. boulardii
sequela, S. torula), Issatchenkia, Kluyveromyces, Pichia,
Wickerhamomyces (e.g., W. anomalus), Starmerella (e.g., S.
bombicola), Mycorrhiza, Mortierella, Phycomyces, Blakeslea,
Thraustochytrium, Phythium, Entomophthora, Aureobasidium pullulans,
Pseudozyma aphidis, Aspergillus and/or Rhizopus spp.
In one embodiment, the microorganism is a killer yeast. As used
herein, "killer yeast" means a strain of yeast characterized by its
secretion of toxic proteins or glycoproteins, to which the strain
itself is immune. The exotoxins secreted by killer yeasts are
capable of killing other strains of yeast, fungi, or bacteria. For
example, microorganisms that can be controlled by killer yeast
include Fusarium and other filamentous fungi. Such yeasts can
include, but are not limited to, Wickerhamomyces (e.g., W.
anomalus), Pichia (e.g., P. anomala, P. guielliermondii, P.
occidentalis, P. kudriavzevii), Hansenula, Saccharomyces,
Hanseniaspora, (e.g., H. uvarum), Ustilago maydis, Debaryomyces
hansenii, Candida, Cryptococcus, Kluyveromyces, Torulopsis,
Ustilago, Williopsis, Zygosaccharomyces (e.g., Z. bailii), and
others.
In one embodiment, the microbe used for the present invention is
Pseudozyma aphidis. Pseudozyma aphidis is an efficient producer of
mannosylerythritol lipids (MELs).
In one embodiment, the microbe can be chosen from strains of killer
yeast. In even more preferred embodiments, the microbes are Pichia
strains, including, for example, P. anomalus (Wickerhamomyces
anomalus), P. kudriavzevii (Wickerhamomyces kudriavzevii), and/or
P. guilliermondii (Meyerozyma guilliermondii).
In one embodiment, the microbial strain is Starmerella bombicola,
which is an efficient producer of sophorolipids (SLPs).
In one embodiment, the microorganisms are bacteria, including
Gram-positive and Gram-negative bacteria. The bacteria may be, for
example Agrobacterium radiobacter, Arthrobacter, Azobacter (e.g.,
A. vinelandii, A. chroococcum), Azospirillum brasiliensis, Bacillus
(e.g., B. subtilis, B. licheniformis, B. firmus, B. laterosporus,
B. megaterium, B. amyloliquifaciens), Clostridium (e.g., C.
butyricum, C. tyrobutyricum, C. acetobutyricum, Clostridium NIPER
7, and C. beijerinckii), Gordonia, Mycobacterium, Nocardia,
Pseudomonas (e.g., P. chlororaphis subsp. aureofaciens (Kluyver),
P. aeruginosa), Ralslonia eulropha, Rhodococcus (e.g., Rhodococcus
erythropolis), Rhodospirillum rubrum, Rhizobium and/or,
Sphingomonas paucimobilis,
In one embodiment, the microbe is a non-pathogenic strain of
Pseudomonas (e.g., P. aeruginosa). Preferably, the strain is a
producer of rhamnolipid biosurfactants (RLP).
In one embodiment, the microbe is Rhodococcus erythropolis, which
is an effective producer of trehalose lipids (TLs).
In one embodiment, the microorganism is a strain of B. subtilis,
such as, for example, B. subtilis var. lotuses B1 or B2, which are
effective producers of, for example, surfactin and iturin. This
specification incorporates by reference International Publication
No. WO 2017/044953 A1 to the extent it is consistent with the
teachings disclosed herein.
In another embodiment, the microorganism is a strain of Bacillus
amyloliquefaciens, which is also an effective producer of
surfactin.
Other microbial strains including, for example, other strains
capable of accumulating significant amounts of, for example,
glycolipid-biosurfactants or lipopeptide-biosurfactants can be used
in accordance with the subject invention. Additional metabolites
useful according to the present invention include mannoprotein,
beta-glucan and other biological amphiphilic molecules that have
bio-emulsifying and surface/interfacial tension-reducing
properties.
Preparation of Microbe-Based Products
One microbe-based product of the subject invention is simply the
fermentation broth containing the microorganism and/or the
microbial metabolites produced by the microorganism and/or any
residual nutrients. The product of fermentation may be used
directly without extraction or purification.
However, extraction and purification can be easily achieved using
standard extraction and/or purification methods or techniques
described in the literature. For example, in certain embodiments,
the microbe-based product comprises simply the by-products of
microbial growth, either in crude or purified form. In particular
embodiments, the by-products are biosurfactants produced by the
microorganisms grown according to the subject invention.
The microbes and/or broth resulting from the microbial growth can
be removed from the growth vessel and transferred via, for example,
piping for immediate use.
In other embodiments, the composition (microbes, broth, or microbes
and broth) can be placed in containers of appropriate size, taking
into consideration, for example, the intended use, the contemplated
method of application, the size of the fermentation tank, and any
mode of transportation from microbe growth facility to the location
of use. Thus, the containers into which the microbe-based
composition is placed may be, for example, from 1 gallon to 1,000
gallons or more. In other embodiments the containers are 2 gallons,
5 gallons, 25 gallons, or larger.
In certain embodiments, the compositions of the subject invention
have advantages over, for example, biosurfactants alone, including
one or more of the following: high concentrations of mannoprotein
as a part of yeast cell wall's outer surface (mannoprotein is a
highly effective bioemulsifier capable of reaching up to an 80%
emulsification index); the presence of biopolymer beta-glucan (an
emulsifier) in yeast cell walls; the presence of biosurfactants in
the culture, which are capable of reducing both surface and
interfacial tension; and the presence of metabolites (e.g., lactic
acid, ethanol, etc.).
Upon harvesting the microbe-based composition from the growth
vessels, further components can be added as the harvested product
is placed into containers and/or piped (or otherwise transported
for use). The additives can be, for example, buffers, carriers,
other microbe-based compositions produced at the same or different
facility, viscosity modifiers, preservatives, nutrients for microbe
growth, tracking agents, solvents, biocides, other microbes and
other ingredients specific for an intended use.
Other suitable additives, which may be contained in the
formulations according to the invention, include substances that
are customarily used for such preparations. Example of such
additives include surfactants, emulsifying agents, lubricants,
buffering agents, solubility controlling agents, pH adjusting
agents, preservatives, stabilizers and ultra-violet light resistant
agents.
In one embodiment, the composition may further comprise buffering
agents including organic and amino acids or their salts. Suitable
buffers include citrate, gluconate, tartarate, malate, acetate,
lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate,
galactarate, glucarate, tartronate, glutamate, glycine, lysine,
glutamine, methionine, cysteine, arginine and a mixture thereof.
Phosphoric and phosphorous acids or their salts may also be used.
Synthetic buffers are suitable to be used but it is preferable to
use natural buffers such as organic and amino acids or their salts
listed above.
In a further embodiment, pH adjusting agents include potassium
hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate,
hydrochloric acid, nitric acid, sulfuric acid or a mixture
thereof.
In one embodiment, additional components such as an aqueous
preparation of a salt as polyprotic acid such as sodium bicarbonate
or carbonate, sodium sulfate, sodium phosphate, sodium biphosphate,
can be included in the formulation.
Advantageously, in accordance with the subject invention, the
microbe-based product may comprise broth in which the microbes were
grown. The product may be, for example, at least, by weight, 1%,
5%, 10%, 25%, 50%, 75%, or 100% broth. The amount of biomass in the
product, by weight, may be, for example, anywhere from 0% to 100%
inclusive of all percentages therebetween.
Optionally, the product can be stored prior to use. The storage
time is preferably short. Thus, the storage time may be less than
60 days, 45 days, 30 days, 20 days, 15 days, 10 days, 7 days, 5
days, 3 days, 2 days, 1 day, or 12 hours. In a preferred
embodiment, if live cells are present in the product, the product
is stored at a cool temperature such as, for example, less than
20.degree. C., 15.degree. C., 10.degree. C., or 5.degree. C. On the
other hand, a biosurfactant composition can typically be stored at
ambient temperatures.
EXAMPLES
A greater understanding of the present invention and of its many
advantages may be had from the following examples, given by way of
illustration. The following examples are illustrative of some of
the methods, applications, embodiments and variants of the present
invention. They are not to be considered as limiting the invention.
Numerous changes and modifications can be made with respect to the
invention.
Example 1--Cultivation of Pichia Species for Use in Production of
Personal Care Compositions
Pichia yeasts can be used to produce a variety of personal care
compositions, as their cell derivatives can eliminate fungus that
contaminates the skin, reduce pathogenic yeasts growing on the skin
and mucosal surfaces, decrease bacterial contamination of the skin
and mucosa, and stimulate the production of collagen and elastin in
skin cells (to name a few).
The basic cultivation medium for producing Pichia anomala
(Wickerhamomyces anomalus), Pichia kudriavzevii and Pichia
guilliermondii (Meyerozyma guilliermondii) is identical for all
three species. The medium comprises 2% glucose, 1% yeast extract,
1% canola oil, 5% glycerol, and 50 mM citrate buffer. If Pichia
guilliermondii is being cultivated for production of chitinase,
0.1% micronized chitin is added. If organic status is desired for
the cosmetic product, no inorganic salts are used, and all nutrient
medium components must be certified for the organic status.
The temperature for fermentation can range from 25-30.degree. C.,
and initial pH can range from 5.5-6.0. Saturated oxygen can range
from 15-25% (of 100% ambient air). Total fermentation time can be
up to 72 hours (determined by reaching the stationary phase).
Once a culture has been produced, it should immediately be cooled
down to 5-10.degree. C. to prevent possible degradation of active
substances in the supernatant. Then the yeast biomass and yeast
supernatant are isolated via centrifugation or microfiltration (or
combination thereof) through a 0.1-micron filter while keeping the
process at a temperature no higher than 10.degree. C.
Protein molecules can be precipitated using a salting technique.
The proteins are salted out by increasing concentration of salt,
i.e., ammonium sulfate. After the proteins are concentrated 10-20
folds (or more, depending on the necessary concentration for the
final personal care product), they are collected and washed out by
a cold saline solution 2-3 times (with constant mixing for 1 hour
each time).
Enzymatic activity of the concentrated product can be stabilized by
mixing it with sodium alginate to a final concentration of 1%
sodium alginate, or mixing it with xanthan gum to a final
concentration of 0.5% of xanthan gum.
The antibacterial or antifungal activity of the final resulting
substrate can be tested. Antifungal capabilities can be tested by
well plate assays with Candida yeasts (of clinical significance)
and Malassezia fungus, whereas for antibacterial activity, the
cultures of E. coli and P. aeruginosa can be used. Growth
inhibition should be measured using the diameter of inhibition in
millimeters around the well.
Once testing has been conducted, the resulting composition can be
used to produce a variety of personal care products, including
cosmetic ointments, creams, shampoos, soaps, bath salts, sanitizing
solutions and oils for hands and feet, facial washes and creams,
and others.
The yeast biomass resulting from these cultivation methods can be
used for preparation of other products, including animal and fish
feed as a source of proteins, phytase, vitamins and minerals, as
well as any other application where live or inactive yeast cells
are useful, e.g., soil amendments for agriculture and horticulture,
and enhanced oil recovery in oil drilling operations.
Example 2--Fermentation of Starmerella bombicola for Sophorolipid
(SLP) Production in a 550 Gallon Reactor
A portable, fully enclosed reactor, designed specifically for yeast
growth and biosurfactant production, is operated by PLC and
comprises water filtration, a temperature control unit, an impeller
and a microsparger. The reactor has a working volume of 500 gallons
when growing S. bombicola for SLP production.
In preferred embodiments, the nutrients for SLP production comprise
glucose, urea, yeast extract, and used vegetable cooking oil.
The reactor is inoculated with 50 liters of liquid culture grown in
another reactor. The duration of the cultivation cycle for SLP
production is 5 days, at 25.degree. C. and pH 3.5. The final
concentration of SLP is roughly 10-15% of working volume,
containing 70-75 gallons of SLP.
The culture can be collected into a separate tank. After SLP is
allowed to settle to the bottom of the tank, it can be removed and
processed as desired. The remaining (approximately) 420 gallons of
culture in the tank can comprise from 3-5 g/L of residual SLP.
Example 3--Methods of Treating a Skin Condition
In one embodiment, the subject invention provides methods of
treating a skin condition, wherein the topical cosmetic composition
is applied directly to an area of the skin, i.e., integument, where
such a condition exists. The composition can be applied, for
example, to the skin of the face, ears, scalp, neck, back
shoulders, arms, hands, chest, stomach, underarms, feet, buttocks,
and legs.
In some embodiments, "applying" the composition can comprise
spreading the composition on the area of skin and leaving the
composition on, and/or rubbing it into, the skin until it is fully
absorbed. In some embodiments, the composition can be applied to
the skin for a certain amount of time and then rinsed from the skin
using, for example, water.
In certain embodiments, the topical cosmetic composition is applied
every other day, once daily, or twice daily. In some embodiments,
the topical composition is applied every other day, once daily, or
twice daily, for an indefinite period of time, e.g., for at least
one, two, three weeks, or longer, in order to achieve and/or
maintain the treatment of the skin condition.
In one embodiment, the composition can be applied to the skin in an
amount from about 0.001 to about 100 mg per cm.sup.2 of skin, more
typically from about 0.01 to about 20 mg/cm.sup.2, or from about
0.1 to about 10 mg/cm.sup.2. More or less may be used, however,
depending upon the size of the area of skin to be treated.
In preferred embodiments, the composition comprises one or more
biosurfactants selected from glycolipids (e.g., SLPs, MELs, TLs and
RLPs) and lipopeptides (e.g., surfactin, iturin and fengycin). In
additional embodiments, the composition preferably comprises
therapeutically effective amounts of enzymes and/or proteins
produced by microorganisms, such as, e.g., exo-beta-1,3-glucanase,
chitinase, esterases, lipases, glycosidases, amylases, and
proteases beneficial for improving skin health.
Other ingredients that are helpful for improving skin health can be
applied with the composition, depending upon the skin condition
being treated.
The topical cosmetic compositions and methods of the subject
invention can be used to treat and/or prevent a variety of skin
conditions, including, for example, age spots (e.g.,
hyperpigmentation), acne, scars, body odor, aging-related
conditions (e.g., wrinkles, looseness and dryness), and scalp
issues (e.g., dandruff, seborrheic dermatitis and hair loss).
Additionally, the topical cosmetic compositions can be used as a
cleanser to remove makeup and other impurities from the face and
skin. The method can be applied according to any of the following
Examples, though it is not intended to be limited thereto.
Example 4--Hyperpigmentation Conditions
In some embodiments, a method is provided for treating age spots or
other hyperpigmentation conditions on a subject's skin, wherein the
topical composition is applied directly to an area of the skin
where such a condition exists for a time sufficient to achieve a
desired reduction of pigmentation. In preferred embodiments, the
topical cosmetic composition comprises a combination of MELs and
SLPs. In one embodiment, the composition is formulated as a lotion,
gel or cream.
In certain embodiments, the composition is applied at least once or
twice daily for at least one, two, three weeks, or longer. In some
embodiments, the topical composition is applied daily for an
indefinite period of time in order to achieve and/or maintain a
level of reduction in pigmentation of the skin.
In certain embodiments, the subject invention provides methods of
treating actinic keratoses, seborrheic keratoses, and other
hyperpigmentation conditions of the skin, including treating,
ameliorating, diminishing the appearance of, or preventing such
conditions, by applying a topical cosmetic composition of the
subject invention to the skin in need of treatment.
In some embodiments, the method is also useful for reducing
otherwise unwanted pigmentation, such as by lightening the skin
overall, as well as by improving the signs of environmental,
age-related, or UV-related skin aging.
As used herein, "hyperpigmentation condition" refers to any
condition or disorder of the skin wherein discoloration or an
abnormally colored growth on the skin occurs. Hyperpigmentation can
include excessive or unwanted pigmentation. The hyperpigmentation
may result from increased presence of one or more of the different
types of melanin biosynthesized in skin and/or follicles and
deposited in hair or skin, relative to a subject's baseline
pigmentation. Factors such as aging, environmental stress, and UV
exposure can be potential causes of the development of
hyperpigmentation. Examples of hyperpigmentation conditions
include, but are not limited to, age spots, liver spots, freckles,
mottled and discrete pigmentation, melasma and the like.
Treatment of hyperpigmentation or hyperpigmented skin or hair
refers to eradicating, reducing, ameliorating, reversing or
preventing one or more of the unwanted features associated with
hyperpigmentation, such as producing a perceptible lightening of
the skin or hair in the affected area. Lightening hyperpigmented
areas of the skin may be desirable, in one embodiment, in
diminishing age spots; lightening a suntan; evening or optimizing
skin tones, e.g., in areas of mottled hyperpigmentation; in
treating melasmic and chloasmic patches, freckles, and post-injury
hyperpigmentation.
In some embodiments of the subject methods, the compositions are
applied directly to a site of hyperpigmentation on the skin (i.e.,
directly onto an age spot). In some embodiments, the compositions
are applied daily to achieve and/or maintain a reduction in
pigmentation in the skin.
The topical composition may remain on the affected area in need of
lightening or may be rinsed off or otherwise removed, depending on
the mode of application.
In certain embodiments, application of the topical cosmetic
composition may be repeated for a time sufficient to achieve a
reduction in pigmentation in the area of application. In order to
maintain the desired effect, the method can be continued for as
long as the effect is desired. This may entail topical application
at least once daily for at least one week, at least two weeks, at
least four weeks, or at least eight weeks or more. Once the
application of the topical composition is discontinued, the desired
reduction in pigmentation may also diminish.
The method may be employed prophylactically to forestall the
development of hyperpigmentation conditions on the skin.
In some embodiments, the compositions and methods are useful for
lightening skin, which includes eradicating, reducing,
ameliorating, and/or reversing a baseline degree of subject
pigmentation. Skin lightening may be measured by observing changes
in Fitzpatrick scale value of a subject. The Fitzpatrick Scale,
Fitzpatrick skin typing test, or Fitzpatrick phototyping scale, is
a numerical classification schema for the color of skin, and
remains a recognized tool for dermatologic research of skin
color.
The Fitzpatrick Scale measures several components, including
Genetic Disposition, Reaction to Sun Exposure and Tanning Habits,
and classifies skin into six types: Type I (scores 0-7) refers to
white, very fair skin, freckles, typical albino skin, that always
burns, never tans; Type II (scores 8-16) refers to white, fair
skin, that usually burns, or tans with difficulty; Type III (scores
17-24) refers to beige, which is very common, and which sometimes
suffers mild burn, gradually tans to a light brown; Type IV (scores
25-30) refers to beige skin with a brown tint, which is typical of
Mediterranean Caucasian skin, and which rarely burns, tans with
ease to a moderate brown; Type V (scores over 30) refers to dark
brown skin which very rarely burns, tans very easily; Type VI
refers to black skin that never burns, tans very easily, and is
deeply pigmented.
In some embodiments of the invention, the treatments are capable of
changing the treated area of skin by at least one or two skin types
on the Fitzpatrick scale. When lightening skin, it may be desirable
to apply the composition over a large area of skin (e.g., over the
entire skin of the face).
Skin for which the subject composition may be useful includes, but
is not limited to, discolored or uneven skin, dark complexions,
hyperpigmented skin, post-injury hyperpigmented skin, dark circles
under the eyes, post-inflammation hyperpigmented skin, skin having
age spots, liver spots, melasma, cholasma or freckles, yellowed
skin, stained nails, or skin, scalp, legs, face, or other areas
where lightening or color reduction are desired.
Specific benefits which may be achieved include, but are not
limited to, reducing pigmentation of dark or hyperpigmented skin;
reducing age spots or liver spots; reducing pigmented birthmarks,
sun damage, tans, and pigmented acne marks; evening out or
optimizing skin discoloration; decreasing the appearance of dark
circles under the eyes; treating melasma, cholasma, freckles,
yellowing of skin, and post-inflammation and post-injury
hyperpigmentation; lightening hair on the scalp, legs, face, and
other areas where whitening and color reduction are desired; and
removing or reducing nail stains.
Example 5--Actinic Keratosis (Using 1% SLP Water and Cream
Solutions)
A water solution of 1% by weight SLP was tested on a flat brown
spot on a human subject's face. Initially, the spot had two large
sub-spots near the right eye, each the size of a dime. After two
months of daily application of the solution, the upper sub-spot
nearly disappeared and the lower sub-spot became significantly
lighter.
A cream composition containing 1% by weight SLP was prepared by
mixing SLP with a commercial moisturizing cream. Treatment was
conducted by applying the cream on three actinic keratosis spots.
The cream was applied twice a day. One of the spots, which began
gibbous, became flatter and lighter in color. Another spot, which
began flat, became lighter and divided into two spots, with a light
skin color in between. The third spot lightened to a light shade of
brown.
Example 6--Acne Vulgaris
In certain embodiments, the subject compositions and methods can be
used for treating a skin condition selected from acne and/or other
blemish-causing conditions. The treatment of acne can comprise
preventing, removing and/or reducing the appearance of acne and/or
other blemishes of the skin.
In one embodiment, the method of treating acne comprises applying a
topical cosmetic composition of the subject invention to a
subject's skin, wherein the topical cosmetic composition comprises
MELs, SLPs or a combination thereof. In one embodiment, the MELs
are added to the composition in an amount of about 0.5% to 2.0% by
weight, preferably about 1.0%. In one embodiment, the SLPs are
added in an amount of about 0.1% to 1% by weight, preferably about
0.5%.
In one embodiment, the composition is formulated as a gel, lotion
or cream. In another embodiment, the composition is formulated as a
cleanser or soap.
The method of treating acne can further comprise applying a
therapeutically effective amounts of a known comedolytic or
anti-acne agent. For example, from about 0.001% to about 5.0% by
weight of hydroxy acid, glycolic acid, lactic acid, salicylic acid,
retinoids, benzoyl peroxide, erythromycin, tretinoin, tazarotene,
azelaic acid, adapalene, dapsone, and clindamycin can be applied.
The salt forms of these compounds may also be utilized.
Symptoms of acne or acne-like conditions include, but are not
limited to, the appearance of various types of skin blemishes
(e.g., lesions, comedones, macules, whiteheads, blackheads,
pustules, papules, nodules and cysts), which can cause symptoms
such as inflammation, pain, redness, swelling, bumps, scarring,
scabbing and infections.
The term "lesion" is generally used to denote an infected or
diseased patch of skin. A lesion can involve an infected sebaceous
gland. Some lesions are more severe than others. Examples of skin
lesions are comedones, macules, papules, pustules, nodules and
cysts. The term "comedo" (plural "comedones") is used to describe a
sebaceous follicle plugged with dirt, other cells, tiny hairs, or
bacteria. Comedones include the so-called "blackheads," which can
also be referred to as "open comedones," which have a spot or a
surface that appears black. Comedones also include slightly
inflamed, skin colored bumps, as well as "whiteheads," which have a
spot or a surface that appears white. The term "macule" generally
refers to a flat spot or area of the skin with a changed color,
such as a red spot. The term "pustule" is generally used to refer
to an inflamed, pus-filled lesion, or a small inflamed elevation of
the skin that is filled with pus. The term "papule" is generally
used to refer to a small, solid, usually inflammatory elevation of
the skin that does not contain pus. The term "nodule" is generally
used to refer to an elevation of a skin that is similar to a papule
but is white and dome-shaped. Colloquially, a papule, a pustule or
a nodule can be referred to as a "pimple" or a "zit." The term
"cyst" generally refers to an abnormal membranous sac containing a
liquid or semi-liquid substance containing white blood cells, dead
cells, and bacteria. Cysts can be painful and extend to deeper
layers of skin.
Depending on severity, acne can be mild, moderate or severe. Mild
acne is generally categorized by the appearance of blackheads and
whiteheads, but can also include papules and pustules. Moderate
acne is generally characterized by appearance of more painful,
deep-rooted, inflamed lesions, which can result in scarring. Severe
acne is characterized by the appearance of deep-rooted inflammatory
lesions, including cysts and nodules, which can be painful and can
produce scarring. Acne conglobate is a category of acne
characterized by highly inflammatory cysts that communicate under
the skin with abscesses and burrowing sinus tracts.
Specifically, treatment of acne or its related symptoms refers to
eradicating, reducing, ameliorating, reversing or preventing one or
more of the unwanted features associated with acne, such as
reducing the redness, pain, inflammation and overall appearance of
comedones in the affected area. Treatment of acne can also lead to
lessening the severity of acne in a patient.
The subject compositions and methods can be used to treat blemishes
caused by mild, moderate, severe and cystic acne. In one
embodiment, the method comprises applying the composition at least
once or twice daily for at least one, two, three weeks or longer.
The composition can be applied daily for an indefinite period of
time in order to achieve and/or maintain a reduction of blemishes
and related symptoms.
Example 7--Skin Replenishment
In certain embodiments, the subject compositions and methods can be
used for replenishing skin. In one embodiment, a method is provided
for replenishing skin in need thereof, by applying a topical
cosmetic composition of the subject invention to the skin, wherein
the composition comprises MELs. In preferred embodiments, the
amount of MELs in the composition is about 0.5% to 2.0% by weight,
preferably about 1.0%. The composition can be formulated as, for
example, a gel, lotion or cream.
As used herein, skin in need of replenishment is skin that is
afflicted with a skin condition selected from dryness, roughness,
fragility, looseness, lack of suppleness, dullness, wrinkles, any
aging-related skin condition or any skin condition caused by
environmental factors, such as, e.g., sun, wind, heat, cold,
makeup, oil, dirt or pollution.
In one embodiment, the method of replenishing skin further
comprises applying a therapeutically effective amount of a known
skin-replenishing compound. For example, from about 0.001 to about
5.0% by weight of hyaluronic acid, ceramides, sodium PCA, glycerin,
glycerol, cholesterol and/or phospholipids can be added, as well as
silicones, petrolatum, triglycerides, omega fatty acids, AHAs
(e.g., glycolic acid) and/or BHA (e.g., salicylic acid).
In one embodiment, the method of replenishing skin can further
comprise applying the protein lactoferrin to the skin.
The method may be employed prophylactically to forestall aging of
skin, including in individuals that have not manifested signs of
skin aging, for example, in individuals under 25 years of age. The
method may also reverse or treat signs of aging once manifested as
is common in individuals over 25 years of age, or to slow the
progression of dermatological aging in such individuals.
Example 8--Scalp and Hair Health
In certain embodiments, the subject compositions and methods can be
used for treating a scalp condition or hair condition. In one
embodiment, a method is provided for treating a scalp and/or hair
condition by applying a topical cosmetic composition of the subject
invention to the scalp and/or the hair, wherein the composition
comprises MELs and acidic-form SLPs. In one embodiment, the amount
of MELs in the composition is about 0.1% to about 1.0% by weight,
preferably about 0.2%. In one embodiment, the amount of acidic-form
SLPs in the composition is about 0.01% to 1.0%, preferably about
0.1%.
As used herein, the terms "scalp condition" and "hair condition"
encompass human and animal conditions, disorders, or diseases
affecting the scalp and/or the hair. Such conditions include, but
are not limited to, dry hair or scalp, thinning hair, brittle hair,
hair loss, male pattern baldness, alopecia, ringwom, seborrheic
eczema, seborrheic dermatitis, cradle cap, acne, psoriasis, head
lice, tricorrhexis nodosa, dandruff, for example, caused by
Malassezia fungi, folliculitis caused by, for example,
Staphylococcus aureus, and others.
In one embodiment, the topical cosmetic composition is formulated
as a shampoo, conditioner, mousse, hair gel, or hair lotion. In one
embodiment, the composition can further comprise certain additives,
including, for example, proteins (e.g., hydrolyzed vegetable
protein, hydrolyzed wheat protein, hydrolyzed milk protein,
hydrolyzed silk and hydrolyzed collagen), vitamins (e.g.,
panthenol, biotin, vitamin E acetate, vitamin A and D palmitate),
moisturizers/humectants (e.g., glycerin, propylene glycol, sodium
PCA, amino acid-based surfactants, and HLA), emollients (e.g.,
esters, isopropyl myristate, decyl oleate, C12-15 alkyl benzoate),
oils (e.g., coconut, jojoba, aloe vera, safflower, almond, argon),
botanicals (e.g., chamomile, aloe, rosemary), and perfumes (e.g.,
lavender, ylang ylang, patchouli), as well as preservatives, dyes,
pH adjusters and chelating agents.
The method can comprise applying about 10 ml, or 2 teaspoons to 2
tablespoons of the composition to the scalp and/or hair (for
example, by rubbing and/or lathering the composition thereon),
allowing the composition to sit for 1 minute to 5 minutes, and then
rinsing the composition with water. In one embodiment, the
composition is rubbed and/or lathered into the scalp and/or hair
without rinsing. The amount of composition applied to the scalp
and/or hair can depend on the length, amount and thickness of the
subject's hair.
Example 9--Body Odor
In certain embodiments, the subject compositions and methods can be
used for treating body odor.
Body odor is typically caused by the presence of Gram-positive
Propionibacteria and Staphylococcus epidermis, which live in the
ducts of the sebaceous glands and produce propionic acid and
isovaleric acid. These compounds can produce undesirable odors.
In one embodiment, a method is provided for treating body odor by
applying a topical cosmetic composition of the subject invention to
the source of the body odor, for example, the skin of the
underarms. The topical cosmetic composition can comprise MELs and
SLPs. In one embodiment, the amount of MELs in the composition can
be from about 0.01% to about 1.0% by weight, preferably about 0.1%.
In one embodiment, the amount of SLPs in the composition can be
from about 0.01% to about 1.0% by weight, preferably about
0.1%.
In one embodiment, the composition is formulated as a liquid, a
stick, a gel, a spray or a wipe.
The composition can further be applied with, for example, carriers
(e.g., propylene glycol, dipropylene glycol and water), gelling
agents (e.g., sodium stearate, sodium palmitate, sodium arachidate,
sodium behenate), clarifying agents (e.g., nonionic surfactants),
fragrances, chelating agents (e.g., disodium or tetrasodium EDTA),
pH adjusters (e.g., aminomethyl propanol, poloxamine, sodium
hydroxide), antioxidants (e.g., BHT) and colorants (e.g., water
and/or alcohol soluble dyes).
Example 10--Scar Reduction
In certain embodiments, the subject compositions and methods can be
used for reducing the appearance of dermal scars, for example,
scars resulting from injury, acne, or surgery, including
plastic/reconstructive surgery. Scars often result from the
overproduction of collagen by modified dermal fibroblasts, or
myofibroblasts. In normal skin, fibroblasts produce collagen
fibers, which align to form a random "basketweave" pattern.
In response to a wound, fibroblasts may be transformed by
inflammatory stimulants into myofibroblasts. Myofibroblasts deposit
collagen in cross-linked patterns, wherein the collagen fibers
align in a single direction parallel to the skin. In addition,
there is greater collagen density and larger fiber size in scars
compared to normal tissue. The abundant myofibroblasts fail to
undergo apoptosis, and continue to circulate the collagen matrix,
produce new collagen, and cause the new skin to stiffen and
contract.
In one embodiment, a method is provided for reducing the appearance
of a scar by applying a topical cosmetic composition of the subject
invention to the scar, wherein the composition comprises MELs and
SLPs, and/or other agents for skin health. In one embodiment, the
composition can be formulated as a gel or a cream. In preferred
embodiments, the composition further comprises Aloe vera extract,
for example, in gel form.
In one embodiment the amount of MELs in the composition is from
about 0.1% to 2.0% by weight, preferably about 1.0%. In one
embodiment, the amount of SLPs in the composition is from about
0.01% to about 1.0% by weight, preferably about 0.5%.
Advantageously, in one embodiment, the subject compositions can
help to dissolve the misaligned collagen matrix that forms a dermal
scar, as well as inhibit the number of myofibroblasts present in
the dermis in order to diminish the appearance of the scar.
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